Our broad objective is to understand molecular mechanisms for replication, processing, and repair of DNA in bacteria and in human cells. Our studies focus principally upon cellular responses to treatments that damage DNA and/or inhibit DNA replication. This research is relevant to an understanding of the molecular basis of hereditary diseases, such as xeroderma pigmentosum (xp), in which DNA repair is defective. 1. DNA repair is being studied in isolate nuclei from UV irradiated xp and WI38 fibroblasts, in which we have shown phage T4 endonuclease V stimulation of repair synthesis. We will examine kinetics of this synthesis, correlation with pyrimidine dimer excision, other xp complementation groups, and other added nucleases. UV irradiated SV40 DNA will be used as probe to study DNA repair in this quasi in vitro system. Intermediates in SV40 DNA replication are being analyzed following UV irradiation of virus-host cell complex using the T4 endo V to detect dimers in parental and daughter DNA strands. 2. The mechanism of DNA replication inhibition in E. coli by nalidixic acid, thymine starvation, UV irradiation, and dnaB at 42 degrees is being studied, with particular emphasis on the possible involvement of the 3'-5' proofreading nucleases in selective degradation of newly-synthesized DNA and in inducible "error-prone" repair. The induction and role of protein X (the putative reacA gene product) is being studied. The fidelity of DNA polymerase III on dimer-containing synthetic DNA templates will be assayed in the presence of protein X and various bacterial extracts. 3. E. coli mutants deficient in dUTPase, cytosine deaminase, N-glycosidases, and endonucleases will be utilized to determine the role of apurinic and apyrimidinic sites in DNA strand breaks and repair replication in normally growing cells. We will further test our hypothesis that strand breaks are an obligatory consequence of transcription--e.g., by examining the effect of inducing the lac operon on the accumulation of strand breaks in an F'lac during thymine starvation. We will also consider the possibility that transcription enhances depurination and indirectly leads to strand breaks.